APP associates with kinesin-1 via JIP1. In JIP1-decicient neurons, the fast velocity and high frequency of anterograde transport of APP cargo are impaired to reduced velocity and lower frequency, respectively. Interaction of JIP1 with KLC via two novel elements in JIP1 plays an important role in efficient APP axonal transport.

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc ␣ , Alc ␤ , and Alc ␥ . The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP ␣-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent ␥-secretase complex, thereby generating "APP p3-like" and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc ␣ , p3-Alc ␤ , and p3-Alc ␥ , whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor ␤-amyloid species A␤42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc ␣ , p3-Alc ␤ , and p3-Alc ␥ were not equivalent, suggesting that one type of ␥-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect ␥-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects. Alcadein (Alc)5 proteins comprise a family of evolutionarily conserved, type I membrane proteins that are predominantly expressed in neuronal tissues. Alc has been independently identified as a binding protein for the neuron-specific adaptor protein X11L (X11-like) (1) and as a postsynaptic Ca 2ϩ -binding protein, where it is known by the name calsyntenin (2). Alc functions as a cargo-receptor for the kinesin-1 motor that mediates anterograde transport of APP (3, 4), and a mutation in a nematode ortholog of the Alc gene is reported to cause a defect in associative learning (5, 6). Thus, Alc plays important roles in vesicular transport at the subcellular level and in learning behavior at the organismal level. Alc exists as four isoforms in mammals: Alc ␣1 (971 amino acids in humans), Alc ␣2 (981 amino acids in humans), Alc ␤ (956 amino acids in humans), and Alc ␥ (955 amino acids in humans) (1). Alc ␣ , Alc ␤ , and Alc ␥ are * This work was supported, in whole or in part, by National Institutes of Health, NIA, Grants R01 AG23611, P01 AG10491, and P50 AG005138 (to S. G.). This work was also supported in part by Grants-in-aid for Scientific Research on Priority Areas 20023001 (to T. S.) from the Ministry of Education, Science, Culture, Sports, and Technology, Japan. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental

Kinesin-1 anterogradely transports vesicles containing cargo proteins when a protein-protein interaction activates it from an inhibited state. The C-terminal cytoplasmic region of kinesin-1 cargo protein Alcadeinα (Alcα) interacts with the KLC1 subunit's tetratricopeptide repeat (TPR) region, activating kinesin-1's association with vesicles and anterograde transport. We found that either of two 10-amino-acid WD motifs in Alcα cytoplasmic region was necessary and sufficient to initiate this activation. An artificial transmembrane protein containing either WD motif induced kinesin-1's vesicular association and anterograde transport in a KLC-dependent manner, even in the normally inhibiting presence of excess KLC1, thus allowing us to analyze the KLC1 TPR-WD functional interaction in detail in vivo. A part of TPR region was dispensable for the WD motifs' activation of kinesin-1 and transport, indicating that only part of the TPR structure is required for this function in vivo. For a different kinesin-1 cargo protein, JIP1, an 11-amino-acid C-terminal region was sufficient to recruit KLC1 to vesicles, but did not activate transport. These observations suggest that structurally different TPR-interacting peptides may have different effects on kinesin-1. This mechanism may partly explain how kinesin-1 can organize the transport of a wide variety of cargo molecules.

Objective The most common pathogenesis for familial Alzheimer's disease (FAD) involves misprocessing (or alternative processing) of the amyloid precursor protein (APP) by γ-secretase due to mutations of the presenilin 1 (PS1) gene. This misprocessing/alternative processing leads to an increase in the ratio of the level of a minor γ-secretase reaction product (Aβ42) to that of the major reaction product (Aβ40). Although no PS1 mutations are present, altered Aβ42/40 ratios are also observed in sporadic Alzheimer's disease (SAD), and these altered ratios apparently reflect deposition of Aβ42 as amyloid. Methods Using immunoprecipitation-mass spectrometry with quantitative accuracy, we analyzed in the cerebrospinal fluid (CSF) of various clinical populations the peptide products generated by processing of not only APP but also an unrelated protein, alcadein (Alc). Alc undergoes metabolism by the identical APP α-secretases and γ-secretases, yielding a fragment that we have named p3-Alcα because of the parallel genesis of p3-Alcα peptides and the p3 fragment of APP. As with Aβ, both major and minor p3-Alcαs are generated. We studied the alternative processing of p3-Alcα in various clinical populations. Results We previously reported that changes in the Aβ42/40 ratio showed covariance in a linear relationship with the levels of p3-Alcα [minor/major] ratio in media conditioned by cells expressing FAD-linked PS1 mutants. Here we studied the speciation of p3-Alcα in the CSF from 3 groups of human subjects (n = 158): elderly nondemented control subjects; mild cognitive impairment (MCI) subjects with a clinical dementia rating (CDR) of 0.5; SAD subjects with CDR of 1.0; and other neurological disease (OND) control subjects. The CSF minor p3-Alcα variant, p3-Alcα38, was elevated (p < 0.05) in MCI subjects or SAD subjects, depending upon whether the data were pooled and analyzed as a single cohort or analyzed individually as 3 separate cohorts. Interpretation These results suggest that some SAD may involve alternative processing of multiple γ-secretase substrates, raising the possibility that the molecular pathogenesis of SAD might involve γ-secretase dysfunction.

This paper provides a bioimage informatics system of detecting and tracking protein molecules, called APP-GFPs, in a live-cell video captured by a fluorescent microscope. Since both processes encounter many difficulties such as many targets, less appearance information, and heavy background noise, we will try to design the system as robust as possible. Specifically, for the detection, a machine learning-based method is employed. For tracking, a method based on a global optimization strategy is newly developed. Experimental results showed that the speed and direction distributions of molecular motion by the proposed system were very similar to that by manual inspection.